Rho-kinase inhibitors

ABSTRACT

Disclosed are compounds and derivatives thereof, their synthesis, and their use as Rho-kinase inhibitors. These compounds are useful for inhibiting tumor growth, treating erectile dysfunction, and treating other indications mediated by Rho-kinase, e.g., coronary heart disease.

FIELD OF THE INVENTION

[0001] The present invention relates to compounds and derivativesthereof, their synthesis, and their use as Rho-kinase inhibitors. Thesecompounds of the present invention are useful for inhibiting tumorgrowth, treating erectile dysfunction, and treating other indicationsmediated by Rho-kinase, e.g., coronary heart disease.

BACKGROUND

[0002] The pathology of a number of human and animal diseases includinghypertension, erectile dysfunction, coronary cerebral circulatoryimpairments, neurodegenerative disorders and cancer can be linkeddirectly to changes in the actin cytoskeleton. These diseases pose aserious unmet medical need. The actin cytoskeleton is composed of ameshwork of actin filaments and actin-binding proteins found in alleukaryotic cells. In smooth muscle cells the assembly and disassembly ofthe actin cytoskeleton is the primary motor force responsible for smoothmuscle contraction and relaxation. In non-muscle cells, dynamicrearrangements of the actin cytoskeleton are responsible for regulatingcell morphology, cell motility, actin stress fiber formation, celladhesion and specialized cellular functions such as neurite retraction,phagocytosis or cytokinesis (Van Aelst, et al. Genes Dev 1997, 11,2295).

[0003] The actin cytoskeleton is controlled by a family of proteins thatare a subset of the Ras superfamily of GTPases. This subset currentlyconsists of RhoA through E and RhoG (refereed to collectively as Rho),Rac 1 and 2, Cdc42Hs and G25K and TC10 isoforms (Mackay, et al. J BiolChem 1998, 273, 20685). These proteins are GTP (guanine nucleotidetriphosphate) binding proteins with intrinsic GTPase activity. They actas molecular switches and cycles between inactive GDP (guaninenucleotide diphosphate) bound and active GTP bound states. Usingbiochemical and genetic manipulations, it has been possible to assignfunctions to each family member. Upon activation the Rho proteinscontrols the formation of actin stress fibers, thick bundles of actinfilaments, and the clustering of integrins at focal adhesion complexes.When activated the Rac proteins control the formation of lamellopodia ormembrane ruffles on the cell surface and Cdc42 controls filopodiaformation. Together this family of proteins plays a critical part in thecontrol of key cellular functions including cell movement, axonalguidance, cytokinesis, and changes in cell morphology, shape andpolarity.

[0004] Depending on the cell type and the activating receptor, the Rhoproteins can control different biological responses. In smooth musclecells, Rho proteins are responsible for the calcium sensitization duringsmooth muscle contraction. In non-smooth muscle cells the Rho GTPasesare responsible for the cellular responses to agonist such aslysophosphatidic acid (LPA), thrombin and thromboxane A₂ (Fukata, et al.Trends Pharcol Sci 2001, 22, 32). Agonist response is coupled throughheterotrimeric G proteins G_(alpha12) or G_(alpha13) (Goetzl, et al.Cancer Res 1999, 59, 4732; Buhl, et al. J Biol Chem 1995, 270, 24631)though other receptors may be involved. Upon activation Rho GTPasesactivate a number of downstream effectors including PIP5-kinase,Rhothekin, Rhophilin, PKN and Rho kinase isoforms ROCK-1/ROKbeta andROCK-1/ROKalpha (Mackay and Hall J Biol Chem 1998, 273, 20685;Aspenstrom Curr Opin Cell Biol 1999, 11, 95; Amano, et al. Exp Cell Res2000, 261, 44).

[0005] Rho kinase was identified as a RhoA interacting protein isolatedfrom bovine brain (Matsui, et al. Embo J 1996, 15, 2208). It is a memberof the myotonic dystrophy family of protein kinase and contains aserine/threonine kinase domain at the amino terminus, a coiled-coildomain in the central region and a Rho interaction domain at the carboxyterminus (Amano, et al. Exp Cell Res 2000, 261, 44). Its kinase activityis enhanced upon binding to GTP-bound RhoA and when introduced intocells, it can reproduce many of the activities of activated RhoA. Insmooth muscle cells Rho kinase mediates calcium sensitization and smoothmuscle contraction and inhibition of Rho kinase blocks 5-HT andphenylephrine agonist induced muscle contraction. When introduced intonon-smooth muscle cells, Rho kinase induces stress fiber formation andis required for the cellular transformation mediated by RhoA (Sahai, etal. Curr Biol 1999, 9, 136). Rho kinase regulates a number of downstreamproteins through phosphorylation, including myosin light chain (Somlyo,et al. J Physiol (Lond) 2000, 522 Pt 2, 177), the myosin light chainphosphatase binding subunit (Fukata, et al. J Cell Biol 1998, 141, 409)and LIM-kinase 2 (Sumi, et al. J Bio Chem 2001, 276, 670).

[0006] Inhibition of Rho kinase activity in animal models hasdemonstrated a number of benefits of Rho kinase inhibitors for thetreatment of human diseases. Several patents have appeared claiming(+)-trans-4-(1-aminoethyl)-1-(pyridin-4-ylaminocarbonyl)cyclohexanedihydrochloride monohydrate (WO-00078351, WO-00057913) and substitutedisoquinolinesulfonyl (EP-00187371) compounds as Rho kinase inhibitorswith activity in animal models. These include models of cardiovasculardiseases such as hypertension (Uehata, et al. Nature 1997, 389, 990),atherosclerosis (Retzer, et al. FEBS Lett 2000, 466, 70), restenosis(Eto, et al. Am J Physiol Heart Circ Physiol 2000, 278, H1744; Negoro,et al. Biochem Biophys Res Commun 1999, 262, 211), cerebral ischeia(Uehata, et al. Nature 1997, 389, 990; Seasholtz, et al. Circ Res 1999,84, 1186; Hitomi, et al. Life Sci 2000, 67, 1929; Yamamoto, et al. JCardiovasc Pharmacol 2000, 35, 203), cerebral vasospasm (Sato, et al.Circ Res 2000, 87, 195; Kim, et al. Neurosurgery 2000, 46, 440), penileerectile dysfunction (Chitaley, et al. Nat Med 2001, 7, 119), centralnervous system disorders such as neuronal degeneration and spinal cordinjury (Hara, et al. J Neurosurg 2000, 93, 94; Toshima, et al. Stroke2000, 31, 2245) and in neoplasias where inhibition of Rho kinase hasbeen shown to inhibit tumor cell growth and metastasis (Itoh, et al. NatMed 1999, 5, 221; Somlyo, et al. Biochem Biophys Res Commun 2000, 269,652), angiogenesis (Uchida, et al. Biochem Biophys Res Commun 2000, 269,633; Gingras, et al. Biochem J 2000, 348 Pt 2, 273), arterial thromboticdisorders such as platelet aggregation (Klages, et al. J Cell Biol 1999,144, 745; Retzer, et al. Cell Signal 2000, 12, 645) and leukocyteaggregation (Kawaguchi, et al. Eur J Pharmacol 2000, 403, 203;Sanchez-Madrid, et al. Embo J 1999, 18, 501), asthma (Setoguchi, et al.Br J Pharmacol 2001, 132, 111; Nakahara, et al. Eur J Pharmacol 2000,389, 103), regulation of intraoccular pressure (Honjo, et al. InvestOphthalmol Vis Sci 2001, 42, 137) and bone resorption (Chellaiah, et al.J Biol Chem 2000, 275, 11993; Zhang, et al. J Cell Sci 1995, 108, 2285).

[0007] The inhibition of Rho kinase activity in patients has benefitsfor controlling cerebral vasospasms and ischemia following subarachnoidhemorrhage (Pharma Japan 1995, 1470, 16).

SUMMARY OF THE INVENTION

[0008] The compounds and their derivatives presented in this inventionare useful as Rho Kinase inhibitors and thus have utilities in thetreatment of hypertension, atherosclerosis, restenosis, cerebralischemia, cerebral vasospasm, neuronal degeneration, spinal cord injury,cancers of the breast, colon, prostate, ovaries, brain and lung andtheir metastases, thrombotic disorders, asthma, glaucoma andosteoporosis.

[0009] In addition, the compounds of the invention are useful to treaterectile dysfunction, i.e., erectile dysfunction mediated by Rho-kinase.Erectile dysfunction can be defined as an inability to obtain or sustainan erection adequate for intercourse, WO 94/28902, U.S. Pat. No.6,103,765 and U.S. Pat. No. 6,124,461.

[0010] The invention involves compounds of the following structures:

[0011] The compounds of the Formula I can be made according to routine,conventional chemical methods, and/or as disclosed below, from startingmaterials which are either commercially available or producibleaccording to routine, conventional chemical methods. Methods for thepreparation of the compounds are given below in the Examples.

[0012] In the following examples, all temperatures are set forthuncorrected in degrees Celsius; and, unless otherwise indicated, allparts and percentages are by weight.

[0013] The entire disclosure of all applications, patents andpublications, cited above or below, and Provisional Application No.60/349,986, filed Jan. 23, 2002, are hereby incorporated by reference.

Abbreviations and Acronyms

[0014] When the following abbreviations are used herein, they have thefollowing meaning: Ac₂O acetic anhydride anhy anhydrous n-BuOH n-butanolt-BuOH t-butanol CD₃OD methanol-d₄ Celite ® diatomaceous earth filteragent, ® Celite Corp. CH₂Cl₂ methylene chloride CI-MS chemicalionization mass spectroscopy conc concentrated dec decomposition DMEdimethoxyethane DMF N,N-dimethylformamide DMSO dimethylsulfoxide ELSDevaporative light scattering detector EtOAc ethyl acetate EtOH ethanol(100%) Et₂O diethyl ether Et₃N triethylamine HPLC high performanceliquid chromatography-electrospray mass ES-MS spectroscopy NMM4-methylmorpholine Ph₃P triphenylphosphine Pd(dppf)Cl₂[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) Pd(PPh₃)₄tetrakis(triphenylphosphine)palladium(0) Pd(OAc)₂ palladium acetateP(O)Cl₃ phosphorous oxychloride RT retention time (HPLC0) rt roomtemperature THF tetrahydrofuran TFA trifluoroacetic acid TLC thin layerchromatography

EXPERIMENTAL EXAMPLES

[0015] All reactions were performed in flame-dried or oven-driedglassware under a positive pressure of dry argon, and were stirredmagnetically unless otherwise indicated. Sensitive liquids and solutionswere transferred via syringe or cannula, and introduced into reactionvessels through rubber septa. Commercial grade reagents and solventswere used without further purification. Thin layer chromatography (TLC)was performed on Analtech UNIPLATE™ pre-coated glass-backed silica gel60 A F-254 250 μm plates. Column chromatography (flash chromatography)was performed on a Biotage system using 32-63 micron, 60 A, silica gelpre-packed cartridges. Proton (¹H) nuclear magnetic resonance (NMR)spectra were measured with a Varian (300 MHz) spectrometer with residualprotonated solvent (CHCl₃ δ 7.26; MeOH δ 3.30; DMSO δ 2.49) as standard.Low-resolution mass spectra (MS) were either obtained as electron impact(El) mass spectra or as fast atom bombardment (FAB) mass spectra.

[0016] The IUPAC name was obtained using the ACD/I Lab Web service.

[0017] A. Preparation of Chloropyrimidine Intermediates

Intermediate A1 Preparation of 2-amino-4-chloro-5,6-dimethyl-pyrimidine

[0018]

[0019] Step 1. Preparation of 2-amino-5,6-dimethyl-4-pyrimidinone

[0020] To a solution of ethyl 2-acetoacetate (6.0 g, 41.6 mmol) andguanidine carbonate (5.6 g, 31.2 mmol) in EtOH (32 mL) was added 12 NHCl (350 μL). The mixture was refluxed for 16 h. After the reaction wascooled to room temperature, the solid was collected by filtration andwashed with EtOH. A solution of the solid in 1 N NaOH was refluxed for 3h. After the reaction was cooled to room temperature, the aqueousmixture was adjusted to pH=5 with concentrated acetic acid. Theresulting precipitate was collected by filtration, washed with water andthen with hexanes, and dried under vacuum. Desired compound (6.34 g,45.6 mmol; 100% yield); ¹H NMR (D₂O; NaOD) δ 1.47 (s, 3H), 1.29-1.30 (m,2H), 1.22 (s, 3H); ES MS [M+H]⁺=140.

[0021] Step 2. Preparation of 2-amino-4-chloro-5,6-dimethyl-pyrimidine

[0022] The product of the previous step (2.0 g, 14.4 mmol) andphosphorus oxychloride (6 mL, 57.5 mmol), was refluxed for 4 h. Thereaction was cooled to rt and poured over ice. The mixture was separatedand the aqueous layer was extracted with chloroform (3×75 mL). Theaqueous mixture was adjusted to pH=9 with concentrated ammoniumhydroxide. The resulting solid product was collected by filtration,washed with water, and dried under vacuum. Desired compound (963 mg, 6.1mmol; 43% yield); mp=212-220° C.; ES MS [M+H]⁺=158; TLC (CH₂Cl₂—MeOH,90:10); R_(f)=0.72.

Intermediate A2 Preparation of 2-amino-4-chloro-6-(4-pyridyl)pyrimidine

[0023]

[0024] Step 1. Preparation of 2-amino-4-hydroxy-6-(4-pyridyl)pyrimidine

[0025] A solution of guanidine carbonate (7.1 g, 39 mmol, 1.5 eq), ethylisonicotinoyl acetate (10 g, 51.76 mmol), and hydrochloric acid (0.75mL, 9.0 mmol) in absolute ethanol (80 mL) was refluxed under argonovernight. The precipitate formed was filtered, washed with ethanol anddried. The solid was then dissolved in 1 N NaOH (100 mL) and refluxedfor 2 h. The reaction mixture was then cooled to room temperature,acidified with glacial acetic acid, and the solid formed was filteredand dried to afford the desired product as a white solid (5.45 g, 56%).¹H-NMR (DMSO-d₆) δ 6.24 (s, 1H), 6.79 (bs, 2H), 7.85 (d, J=5.1 Hz, 2H),8.62 (d, J=5.3 Hz, 2H), 11.22 (bs, 1H).

[0026] Step 2. Preparation of 2-amino-4-chloro-6-(4-pyridyl)pyrimidine:

[0027] A solution of 2-amino-4-hydroxy-6-(4-pyridyl)pyrimidine (5.45 g,29 mmol) in POCl₃ (12 mL) was refluxed under argon for 5 h. The reactionmixture was cooled to room temperature, poured over ice, and allowed tostir at room temperature for 2 h to ensure the quenching of POCl₃. Atthis time, the mixture was made basic upon addition of 1 N NaOH and thebrown solid was filtered to afford 4.52 g of crude product, which wasused without further purification (NMR analysis showed 1:1product/starting material). The filtrate formed more solid upon standingat room temperature (1 g, NMR analysis showed 2:1 product/startingmaterial). ¹H-NMR (DMSO-d₆) δ 7.34 (bs, 2H), 7.38 (s, 1H), 7.99 (d,J=4.2 Hz, 2H), 8.72 (d, J=4.6 Hz, 2H).

Intermediate A3 Preparation of 2-amino-4-chloro-6-(2-thienyl)pyrimidine

[0028]

[0029] Step 1. Preparation of ethyl-2-(thiophene-2-oyl)acetate.

[0030] A solution of thiophene-2-carboxylic acid (8.9 g, 68.5 mmol),2,2-dimethyl-1,3-dioxane-4,6-dione (12.0 g, 81.6 mmol), and4-dimethylaminopyridine (17.0 g, 138 mmol) in dry CH₂Cl₂ (100 mL) wascooled to 0° C. and treated with a solution of1,3-dicyclohexylcarbodiimide (75 mL, 1.0 M in CH₂Cl₂, 75 mmol). Thereaction was allowed to stir at room temperature for 2 h and thedicyclohexylurea was then filtered and washed with CH₂Cl₂. The filtratewas concentrated at reduced pressure and the residue was dissolved inabsolute ethanol (400 mL). The solution was then treated with a solutionof p-toluenesulfonic acid monohydate (32 g, 168 mmol) in absoluteethanol (100 mL) and refluxed under argon for 1 h. At this time, theethanol was removed at reduced pressure and the residue was dissolved inEtOAc and washed sequentially with H₂O (300 mL), saturated NaHCO₃ (200mL), 1 N HCl (200 mL), saturated NaCl, and dried (MgSO₄). The solventwas removed at reduced pressure and the residue was filtered through apad of silica with 10% EtOAc/90% hexanes to afford the desired productas an oil (13 g, 96%). TLC (20% EtOAc/80% hexane) R_(f) 0.21; ¹H-NMR(DMSO-d₆) δ 1.17 (t, J=7.01, 3H), 4.06-4.14 (m, 4H), 7.25 (t, J=5.1 Hz,1H), 7.98 (d, J=3.8 Hz, 1H), 8.06 (d, J=4.9 Hz, 1H).

[0031] Step 2. Preparation of 2-amino-4-hydroxy-6-(2-thienyl)pyrimidine.

[0032] The procedure was similar to that used for Intermediate A2, step1, using ethyl-2-(thienyl-2-oyl)acetate as starting material. (43%yield). TLC (6% MeOH/94% CH₂Cl₂) R_(f) 0.23; MS ES 194 [M+H]⁺; ¹H-NMR(DMSO-d₆) δ 6.06 (s, 1H), 6.70 (bs, 2H), 7.11 (t, J=4.9 Hz, 1H), 7.64(d, J=4.9 Hz, 1H), 7.70 (d, J=3.6 Hz, 1H), 10.95 (bs, 1H).

[0033] Step 3. Preparation of 2-amino-4-chloro-6-(2-thienyl)pyrimidine.

[0034] The procedure was similar to that of Intermediate A2, step 2,using 2-amino-4-hydroxy-6-(2-thiophene)pyrimidine as starting material.It afforded 33% yield after purification on silica with 15% EtOAc/85%hexanes. TLC (20% EtOAc/80% hexanes) R_(f) 0.29; ¹H-NMR (DMSO-d₆) δ7.16-7.23 (m, 4H), 7.77 (dd, J=0.8, 5.0 Hz, 1H), 7.98 (dd, J=1.0, 3.8Hz, 1H).

Intermediate A4 Preparation of 2-amino-4-chloro-6-(2-furyl)pyrimidine

[0035]

[0036] Step 1. Preparation of 2-amino-4-hydroxy-6-(2-furyl)pyrimidine.

[0037] The general procedure for the preparation of Intermediate A2,(step 1) was used; (37% yield). MS (ES) 178 [M+H]⁺.

[0038] Step 2. Preparation of 2-amino-4-chloro-6-(2-furyl)pyrimidine.

[0039] A solution of 2-amino-4-hydroxy-6-(2-furyl)pyrimidine (1.40 g,7.9 mmol) in POCl₃ (4 mL) was refluxed under argon for 2 h. The POCl₃was distilled; the residue was diluted with EtOAc and poured over icedsaturated NaHCO₃. The layers were separated and the aqueous wasextracted with EtOAc (100 mL). The combined extracts was washed withsaturated NaCl, dried (MgSO₄), and the solvent removed at reducedpressure to afford 0.5 g of crude product, which was used withoutfurther purification. TLC (20% EtOAc/80% hexane) R_(f) 0.26; ¹H-NMR(DMSO-d₆) δ 6.68 (dd, J=1.7, 3.4 Hz, 1H), 6.94 (s, 1H), 7.25 (dd, J=1,3.7 Hz, 1H), 7.91 (dd, J=0.8, 1.9 Hz, 1H).

Intermediate A5 Preparation of6-benzyl-4-chloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-amine

[0040]

[0041] Step 1. Preparation of2-amino-7-benzyl-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidin-4(3H)-one.

[0042] To EtOH (16 mL) cooled to 0° C. (ice/H₂O bath) was added Naspheres (204 mg, 8.9 mmol). The mixture was stirred until all Nadissolved. Methyl 1-benzyl-4-oxo-3-piperidine-carboxylate hydrochloride(3.0 g, 10.1 mmol) and guanidine carbonate (1.4 g, 7.6 mmol) were added.The mixture was refluxed for 16 h. After the reaction was cooled to roomtemperature, the solid was collected by filtration, washed with EtOH,and dried under vacuum. Desired compound (2.58 g, 10.0 mmol; 99+%yield); mp=202-212° (dec.); ES MS [M+H]⁺=257; TLC (CH₂Cl₂—MeOH, 90:10);R_(f)=0.20.

[0043] Step 2. Preparation of6-benzyl-4-chloro-5,6,7,8-tetrahydropyrido[4,3-d]pyrimidin-2-amine.

[0044] A solution of the product from step 1 (3.5 g, 13.7 mmol) in POCl₃(52 mL) was refluxed under argon for 5 h. The reaction mixture wascooled to room temperature, poured over ice, and allowed to stir at roomtemperature for 2 h to ensure the quenching of POCl₃. At this time, themixture was made basic upon addition of ammonium hydroxide and wasextracted with CH₂Cl₂ (3×200 mL). The combined organics were washed with1N NaOH followed by brine, dried (MgSO₄), and concentrated under reducedpressure. The residue was taken up in benzene and was made acidic uponthe addition of 1N HCl in diethyl ether. The brown solid was filtered toafford 0.35 g of crude product, which was used without furtherpurification. ES MS [M+H]⁺=275.

Intermediate A6 Preparation of 2-amino-6-(trifluoromethyl)-4-pyrimidinyl4-methylbenzenesulfonate

[0045]

[0046] To a solution of 2-amino-6-(trifluoromethyl)-4(3H)-pyrimidinone(250 mg, 1.4 mmol), triethylamine (196 μL, 1.4 mmol),N,N-dimethylaminopyridine (17 mg, 0.14 mmol), in CH₂Cl₂ (13 mL) cooledto 0° C. was added p-toluenesulfonyl chloride (534 mg, 2.8 mmol). Themixture was stirred at room temperature for 16 h. The mixture wasdiluted with CH₂Cl₂, washed with H₂O (2×20 mL) followed by brine, dried(Na₂SO₄), evaporated, and dried under vacuum. Desired compound (466 mg,1.4 mmol; 99+% yield; ES MS [M+H]⁺=140.

[0047] Using the above methods for the preparation of A1-A6 andsubstituting the appropriate starting materials, the followingpyrimidine intermediates were also prepared. TABLE 1 ChloropyrimidineIntermediates A

Intermediate No. R₁ R₂ R₃ Physical Data  A7 Me H NH₂ Aldrich  A8 Et HNH₂ Aldrich or Lancaster  A9 H H NH₂ Aldrich A10 t-Bu H NH₂ mp =109-113° C.; ES MS [M + H]⁺ = 186; TLC (90:10 CH₂Cl₂/MeOH); R_(f) =0.37. A11 Me Cl NH₂ Aldrich or Lancaster? A12 —(CH)₄— NH₂ ¹HNMR(DMSO-d₆)δ6.60(s, 2H), 2.55−2.29 (m, 4H), 1.68-1.56(m, 4H) A13—(CH)₅— NH₂ ¹H NMR(DMSO-d₆)δ6.65(s, 2H), 2.72−2.58 (m, 4H), 1.74-1.60(m,2H), 1.55-1.34(m, 4H) A14 —(CH)₃— NH₂ ¹H NMR(DMSO-d₆)δ6.73(s, 2H),2.72−2.57 (m, 4H), 1.89(sept, J=7.0, 2H) A15 i-Pr H NH₂ mp = 104-112°C.; ¹H NMR(D₂O)δ6.11(s, 1H), 2.23−2.11(m, 1H), 0.46(d, J=6.2Hz, 6H); ESMS [M + H]⁺ = 172 A16 CH₃ H Ph-NH— A17 Ph H NH₂ A18 3-pyridyl H NH₂ A192-pyridyl H NH₂ A20 3-NO₂-Ph H NH₂ A21 Cl H NH₂ Aldrich

[0048] B. Preparation of Arylamine Intermediates

Intermediate B1 Preparation of 1-(4-pyridinyl)-1H-indol-5-amine

[0049]

[0050] Step 1. Preparation of 5-nitro-1-(4-pyridinyl)-1H-indole

[0051] To a solution of 5-nitroindole (7.0 g, 43.2 mmol) and4-chloropyridine hydrochloride (7.8 g, 51.8 mmol) in DMF (43 mL) wasadded potassium tert-butoxide (12.1 g, 108.0 mmol), portionwise. Thereaction was heated at 100° C. for 48 h. The mixture was allowed to coolto room temperature and poured into water (400 mL). The resulting solidwas removed by filtration and dried under vacuum. Desired compound (6.04g, 25.3 mmol; 58% yield); ¹H NMR (DMSO-d₆) δ 8.76 (dd, J=1.7, 4.5, 2H),8.68 (d, J=2.2, 1H), 8.06-8.13 (m, 2H), 7.92 (d, J=9.2, 1H), 7.75 (dd,J=1.5, 4.6, 2H), 7.07 (dd, J=0.9, 3.5, 1H); ES MS [M+H]⁺=240.

[0052] Step 2. Preparation of 1-(4-pyridinyl)-1H-indol-5-amine

[0053] A mixture of the product from step 1 (8.27 g, 34.6 mmol) and 10%palladium-on-charcoal catalyst (827 mg) in ethyl acetate (166 mL) andEtOH (9 mL) was stirred under hydrogen at atmospheric pressure for 48 h.Further catalyst (414 mg) was added and the reaction was stirred for 24h. Again, further catalyst (414 mg) was added and the reaction wasstirred an additional 24 h. The catalyst was removed by filtrationthrough diatomaceous earth and the solvent removed from the filtrate byevaporation. The residue was triturated with ether, collected byfiltration, and dried under vacuum. Desired compound (4.67 g, 22.3 mmol;65% yield); mp=149-154° C.; ES MS [M+H]⁺=210; TLC (CH₂Cl₂-MeOH, 95:5);R_(f)=0.29.

Intermediate B2 Preparation of 4-[(4-aminophenyl)sulfanyl]phenol

[0054]

[0055] Step 1. Preparation of 4-[(4-nitrophenyl)sulfanyl]phenol.

[0056] To a solution of nitrobenzenesulfonyl chloride (4 g, 21 mmol) inether (25 mL) was added phenol (1.97 g, 20 mmol) as a solution in ether(25 mL). After being stirred for 15 h at rt, the mixture wasconcentrated to afford a crude solid which was recrystallized fromacetic acid. Desired compound (4.0 g, 16.2 mmol, 76% yield). TLC(Hexanes/EtOAc, 70:30); R_(f)=0.54.

[0057] Step 2. Preparation of 4-[(4-aminophenyl)sulfanyl]phenol.

[0058] To a solution of the product of step 1 (4 g, 16.2 mmol) in EtOH(500 mL) was added SnCl₂.2H₂O (18.3 g, 81 mmol) The solution was warmedto reflux. After being stirred for 3 h, the mixture was allowed to coolto rt, and the volatiles were removed by rotary evaporation. Theresultant slurry was suspended in EtOAc, and solid NaHCO₃ was added.Subsequently, the mixture was filtered, and the filtered solid waswashed thoroughly with EtOAc. The organic filtrate was washed withwater, and the aqueous washes were extracted with EtOAc. The combinedorganic extracts were washed with brine, dried (MgSO₄), filtered, andconcentrated to afford an orange solid, which was used withoutadditional purification. Desired compound (3.0 g, 13.8 mmol, 86% yield).

[0059] TLC (Hexanes/EtOAc, 70:30); R_(f)=0.34.

Intermediate B3 Preparation of(3-aminophenyl)[4-(methylsulfanyl)phenyl]methanone

[0060]

[0061] Step 1. Preparation of[4-(methylsulfanyl)phenyl](3-nitrophenyl)methanone

[0062] 3-nitrobenzoylchloride (5.0 g, 26.94 mmol) was added to asolution of thioanisole (3.16 ml, 26.94 mmol) and 1,2-dichlorethane (95mL). The resulting reaction mixture was cooled to 0° C. (ice/H₂O bath)and 0.5 equivalents of aluminum trichloride (1.8 g, 13.47 mmol) wasadded. The reaction was allowed to stir for 15 min at this temperatureand the cold bath was removed followed by addition of the remainingequivalents of AlCl₃ (2.51 g, 18.87). The reaction solution turned adark greenish/yellow and was allowed to stir at room temp. for 18 h,after which time the reaction was quenched slowly with H₂O (50 mL). Themixture was diluted with CH₂Cl₂ (50 mL) and washed with H₂O (3×50 mL),and the combined organic phases were washed with satd NaHCO₃ (50 mL),dried (MgSO₄) and concentrated under reduced pressure. The crudematerial was purified by silica gel column chromatography (EtOAc/hexane,¼) to afford 3.3 g (44%) of4-(methylsulfanyl)phenyl](3-nitrophenyl)methanone as a solid. EI-LRMSm/z 274 (M⁺); TLC Rf 0.68 (EtOAc/Hex, ⅔).

[0063] Step 2. Preparation of(3-aminophenyl)[4-(methylsulfanyl)phenyl]methanone

[0064] Prepared analogously to Intermediate B2, step 2. The crudeproduct was purified by flash column chromatography, eluting with 70:30Hexanes/EtOAc. TLC: (Hexanes/EtOAc, 70:30); R_(f)=0.15.

Intermediate B4 Preparation of 4-(4-aminophenoxy)phenol

[0065]

[0066] Step 1. Preparation of 4-(4-nitrophenoxy)phenol

[0067] A mixture of p-nitrofluorobenzene (25 g, 0.177 mol),dihydroquinone (19.5 g, 0.177 mol), and sodium hydroxide (7.08 g, 0.177mol) in EtOH/H₂O (1:1 v/v, 176 mL) was heated at reflux for 20 h, andsubsequently allowed to cool to room temperature. The mixture wasfiltered, the filtrate was made acidic with dilute aqueous HCl, and theresultant precipitate filtered to afford the crude product as a yellowsolid. The desired product was recrystallized from EtOH. (15 g, 0.064mol, 37% yield). TLC (Hexanes/EtOAc, 70:30); R_(f)=0.44.

[0068] Step 2. Preparation of 4-(4-aminophenoxy)phenol

[0069] To a solution of the product of step 1 in EtOH (100 mL) was added10% palladium on carbon (200 mg). After being stirred under anatmosphere of hydrogen overnight, the mixture was filtered throughCelite®. The volatiles were removed from the filtrate to provide thecrude product which was purified by flash column chromatography elutingwith Hexanes/EtOAc (85:15, followed by 75:25). Desired product (1.5 g,7.45 mmol, 86%). TLC (Hexanes/EtOAc, 70:30); R_(f)=0.41.

Intermediate B5 Preparation of 4-(4-pyridinylthio)aniline

[0070]

[0071] To a solution of 4-aminothiophenol (20.2 g, 156.5 mmol) inanhydrous DMF (200 mL) was added 4-chloropyridine hydrochloride (24.4 g,161.0 mmol) followed by potassium carbonate (44 g, 318.4 mmol). Thereaction mixture was heated at 80° C. overnight, then diluted with ethylacetate (400 mL) and water (400 mL). The aqueous layer wasback-extracted with ethyl acetate (2×200 mL). The combined organiclayers were washed with a saturated aqueous NaCl solution (200 mL),dried over anhy MgSO₄, and concentrated under reduced pressure. Theresidue was filtered through a pad of silica with ethyl acetate and theresulting material was triturated with an ethyl ether/hexane solution toafford the desired product (24.7 g, 78%). TLC (50% ethyl acetate/50%hexane) R_(f) 0.25; ¹H-NMR (DMSO-d₆) δ 5.67 (bs, 2H), 6.65 (d, J=8.4 Hz,2H), 6.88 (d, J=6.2 Hz, 2H), 7.19 (d, J=8.4 Hz, 2H), 8.27 (d, J=6.2 Hz,2H), MS[M+H]⁺=203.

Intermediate B6 Preparation of 4-[2-(4-pyridinyl)ethyl]aniline

[0072]

[0073] Step 1. Preparation of 4-[(E)-2-(4-nitrophenyl)ethenyl]pyridine

[0074] To an oven dried 500 mL 3-necked flask was added(4-nitrobenzyl)triphenylphosphonium bromide (15 g, 30.42 mmol) followedby the addition of THF (100 mL). The solution was cooled to 0° C. in anice bath. Potassium t-butoxide (3.9 g, 33.02 mmol) was then added in oneportion resulting in an orange suspension. The suspension was maintainedat 0° C. while a solution of 4-pyridine-2-carboxaldehyde (2.7 g, 24.70mmol) in THF (20 mL) was added in 10 minutes. The ice bath was removedand the reaction was stirred at room temperature for 2 h. At this time,the reaction was quenched with saturated ammonium chloride solution (50mL) and stirred for 15 minutes. The mixture was then extracted withethyl acetate (2×100 mL), the combined extracts was washed withsaturated aqueous NaCl solution (100 mL) and dried (MgSO₄). The solventwas removed at reduced pressure and the residue was chromatographed onsilica with 0-50% ethyl acetate in hexanes to afford the desired product(1.8 g, 32%). TLC (50% ethyl acetate/50% hexane) R_(f) 0.28; ¹H-NMR(DMSO-d₆) δ 6.84 (d, J=12.4 Hz, 1H), 6.96 (d, J=12.4 Hz, 1H), 7.14 (d,J=6.2 Hz, 2H), 7.45 (d, J=8.7 Hz, 2H), 8.15 (d, J=8.7 Hz, 2H), 8.47 (d,J=6.2 Hz, 2H).

[0075] Step 2. Preparation of 4-[2-(4-pyridinyl)ethyl]aniline

[0076] To a dry 50 mL flask flushed with argon was added 10% Pd oncarbon (285 mg) followed by the addition of ethanol (12 mL) and theproduct from step 1 (1.8 g, 8.0 mmol). At this time, the argon line wasreplaced with a hydrogen balloon and the reaction was stirred overnight.The mixture was filtered through a pad of Celite® and the filtrate wasconcentrated at reduced pressure. The solid residue was triturated withethyl ether/hexanes to afford the desired product (1.2 g, 67%). TLC (4%acetone/96% methylene chloride) R_(f) 0.09; ¹H-NMR (DMSO-d₆) δ 2.67-2.83(m, 4H), 4.83 (bs, 2H), 6.45 (d, J=8.2 Hz, 2H), 6.84 (d, J=8.2 Hz, 2H),7.20 (d, J=6 Hz, 2H), 8.41 (d, J=6 Hz, 2H).

Intermediate B7 Preparation of 3-fluoro-4-(4-pyridinylsulfanyl)aniline

[0077]

[0078] Step 1. Preparation of4-[(2-fluoro-4-nitrophenyl)sulfanyl]pyridine.

[0079] A solution of 4-mercaptopyridine (4.2 g, 35.6 mmol),3,4-difluoronitrobenzene (5.7 g, 35.7 mmol), and potassium carbonate(12.4 g, 89.7 mmol) in anhydrous DMF (40 mL) was stirred at 40° C. andunder argon for 3 h. TLC showed complete reaction. The mixture wasdiluted with ethyl acetate (100 mL) and water (100 mL) and the aqueouslayer was back-extracted with ethylacetate (2×100 mL). The organiclayers were washed with a saturated NaCl solution (100 mL), dried(MgSO₄), and concentrated under reduced pressure. The crude product waspurified by column chromatography with 50% ethyl acetate/50% hexanes. Itafforded the desired product as a yellow solid (6.3 g, 71%). TLC (50%EtOAc/50% hexane) R_(f) 0.53; ¹H-NMR (DMSO-d₆) δ 7.27 (dd, J=0.76, 4.2Hz, 2H), 7.78 (dt, J=0.76, 7.2 Hz, 1H), 8.11-8.15 (m, 1H), 8.28-8.33 (m,1H), 8.5 (dd, J=1.4, 4.6 Hz, 2H), MS [M+H]⁺=251.

[0080] Step 2. Preparation of 3-fluoro-4-(4-pyridinylsulfanyl)aniline.

[0081] A slurry of 3-fluoro-4-pyridinylthio)nitrobenzene (6.3 g, 25.2mmol), iron powder (6.0 g, 107.4 mmol), acetic acid (100 mL), and water(1 mL) were stirred at room temperature overnight. The mixture wasdiluted with Et₂O (100 mL) and water (100 mL). The aqueous phase wasadjusted to pH 5 with a 4 N NaOH solution. The combined organic layerswere washed with an aqueous saturated NaCl solution (100 mL), dried(MgSO₄), and concentrated under reduced pressure. The residue waspurified by column chromatography with 50% ethyl acetate/50% hexanes. Itafforded the desired product as a white solid (4.8 g, 86%). TLC (50%EtOAc/50% hexane) R_(f) 0.28; ¹H-NMR (DMSO-d₆) δ 6.04 (bs, 2H),6.47-6.51 (m, 2H), 6.91 (d, J=6.1 Hz, 2H), 7.22 (t, J=8.4 Hz, 1H), 8.30(d, J=6.4 Hz, 2H).

[0082] Using similar methods to those described for the preparation ofIntermediates B1-B7, the following additional compounds were alsoprepared: TABLE 2 Arylamine Intermediates B

Intermediate No. Z (R₅)_(n) X A Physical Properties B8 CH H (4)-S—CH₂—pyrid-4-yl TLC Rf = 0.12(50% EtOAc/50% hexanes). 1H NMR(DMSO-d6)δ3.91(s,2H), 5.26(bs, 2H), 6.44(d, J=8.7Hz, 2H), 6.96(d, J=8.7 Hz, 2H), 7.12(d,J=6.3Hz, 2H), 8.40 (d, J=6.0Hz, 2H). B9 CH 3-CF₃ (4)-S— pyrid-4-yl TLCRf = 0.10(50% EtOAc/50% hexanes). 1H NMR(DMSO-d6)δ6.21(bs, 2H), 6.84-6.87(m, 3H), 7.10(d, J=2.4Hz, 1H), 7.39 (d, J=8.4Hz, 1H), 8.29(d,J=6.3Hz, 2H). B10 CH H (4)-O— isoquinolin-5-yl B11 CH 3-F (4)-O—isoquinolin-5-yl 1H NMR(DMSO-d6)δ5.42(bs, 2H), 6.41- 6.55(m, 2H),6.81-7.05(m, 2H), 7.48-7.54 (m, 1H), 7.73-7.76(m, 1H), 8.06-8.08(m, 1H),8.54-8.56(m, 1H), 9.32(s, 1H). B12 CH 3,5-(Cl)₂ (4)-O— isoquinolin-5-ylTLC Rf = 0.29(45% EtOAc/55% hexanes). 1H NMR(DMSO-d6)δ5.73(bs, 2H), 6.69(dd, J=1.1 and 8.0Hz, 1H), 6.75(s, 2H), 7.51(t, J=7.7Hz, 1H), 7.78(d,J=8.2Hz, 1H), 8.12(d, J=5.9Hz, 1H), 8.58(d, J=5.6 Hz, 1H), 9.34(bs, 1H).B13 CH H (4)-S— pyrid-4-yl TLC Rf = 0.07(100% EtOAc). 1H NMR(DMSO-d6)δ5.84(bs, 2H), 6.95-6.99(m, 3H), 7.32(d, J=8.6Hz, 1H), 8.00(d,J=2.8 Hz, 1H), 8.31(d, J=4.7Hz, 2H). B14 CH 3,5-(Cl)₂ (4)-S— pyrid-4-yl1H NMR(DMSO-d6)δ6.30(bs, 2H), 6.82(s, 2H), 6.89, (d, J=6.0Hz, 2H),8.33(d, J=6.1 Hz, 2H). B15 CH 2,5-(F)₂ (4)-S— pyrid-4-yl B16 CH 3-Cl(4)-S— pyrid-4-yl B17 CH H (4)-S— isoquinolin-5-yl B18 CH H (4)-CH₂—S—pyrid-4-yl B19 CH H (4)-S— pyrid-3-yl B20 CH H (3)-S— pyrid-4-yl B21 CHH (4)-O— quinolin-5-yl

[0083] C. Preparation of Examples of the Invention

Example 1 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0084]

[0085] A suspension of 2-amino-4-chloro-6-methylpyrimidine (IntermediateA7, 0.2 g, 1.3 mmol), 3-fluoro-4-(4-pyridinylthio)aniline (IntermediateB7, 0.3 g, 1.3 mmol), and K₂CO₃ (0.2 g, 1.3 mmol) in o-xylene (1.3 mL)was heated to 100° C. in a 5 mL reaction-vial overnight. The reactionmixture was diluted with MeOH and coated on silica and purified by MPLC(Biotage) with 5-7% MeOH in CH₂Cl₂. It afforded 74 mg of product (18%yield). TLC (6% MeOH/94% CH₂Cl₂) R_(f) 0.29; MS ES 328 [M+H]⁺; ¹H-NMR(DMSO-d₆) δ 2.12 (s, 3H), 5.92 (s, 1H), 6.38 (bs, 2H), 6.96 (d, J=5.1Hz, 2H), 7.39-7.52 (m, 2H), 8.26 (d, J=11.9 Hz, 1H), 8.33 (d, J=4.8 Hz,2H), 9.55 (bs, 1H).

Example 2 Preparation of6-ethyl-N4-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]-2,4-pyrimidinediamine

[0086]

[0087] 2-Amino-4-chloro-6-ethylpyrimidine (Intermediate A8, 55.1 mg,0.25 mmol) and Intermediate B7(39.4 mg, 0.25 mmol) were suspended in0.01 M aqueous HCl (500 μL). The mixture was refluxed for 6 h. Thereaction was cooled to room temperature and the solvent was evaporatedby vacuum. The residue was purified by reversed phase chromatography ona YMC Pack-pro C18 column (trademark) eluting with acetonitrile/H₂O(10:90-90:10 gradient). The compound was further purified by preparativeTLC eluting with CH₂Cl₂-MeOH (90:10). Desired compound (2.9 mg, 0.0085mmol; 34% yield); ¹H NMR (Methanol-d₄) 8.16 (dd, J=1.7, 4.7, 2H),8.00-8.04 (m, 1H), 7.37 (m, 2H), 6.93 (dd, J=1.8, 4.9, 2H), 5.91 (s,1H), 2.39 (q, J=7.7, 2H), 1.13 (t, J=7.5, 3H); ES MS [M+H]⁺342; TLC(CH₂Cl₂-MeOH, 90:10); R_(f)=0.48.

Examples 3-26

[0088] Using the above procedures, the following examples of pyridineswere synthesized and are summarized in Table 3. TABLE 3

a.

b.

c.

d.

e.

f.

g.

h.

i.

Intermediate Intermediate Pyrimidone Amine Ex. No. (A) (B) R₁ R₂ R₄Analytical Data  3 A9 B1 H H a mp = 245-247° C.; ¹H NMR(Methanol-d₄)8.79(d, J=5.7, 2H), 8.32(s, 1H), 8.13 (d, J= 5.9, 2H), 7.97(d, J=9.2,1H), 7.88(d, J = 3.6, 1H), 7.71(d, J=7.3, 1H), 7.56(d, J= 9.1, 1H),6.94(d, J=4.0, 1H), 6.33(d, J= 7.6, 1H); ES MS [M + H]⁺= 303.  4 A1 B1CH₃CH₂— H a mp = 230-233° C.; ¹H NMR(DMSO-d₆) 12.43(s, 1H), 10.51(s,1H), 8.79(d, J=6.3, 2H), 8.22(s, 1H), 7.87-8.23(m, 5H), 7.46 (d, J=8.4,1H), 6.85(d, J=3.3, 1H), 6.14(s, 1H), 2.51-2.61(m, 2H), 1.19(t, J=7.5,3H); ES MS [M + H]⁺ = 331.  5 A1 B1 CH₃— Cl a mp = 238-241° C.; ES MS[M + H]⁺ = 351; TLC: R_(f) = 0.71(CH₂Cl₂—MeOH, 95:5).  6 A1 B1 —(CH₂)₄—a ¹H NMR(DMSO-d₆)11.75(s, 1H), 10.59(s, 1H), 8.78(d, J=5.4, 2H), 8.21(s,1H), 7.87- 7.97(m, 5H), 7.47(d, J=8.1, 1H), 6.85 (d, J=3.4, 1H), 6.21(s,1H), 1.29(s, 9H); ES MS [M + H]⁺ = 359.  7 A1 B1 —(CH₂)₃)₃— a ¹HNMR(Methanol-d₄)8.80 (d, J 6.4, 1H), 8.21(d, J=6.6, 1H), 8.16(s, 1H),7.99 (d, J=8.9, 1H), 7.90(d, J=3.3, 1H), 7.60 (d, J=8.9, IH), 6.96(d,J=4.0, 1H), 2.93- 2.99(m, 2H), 2.86(s, 2H), 2.23-2.29(m, 2H); ES MS [M +H]⁺ = 343; TLC: R_(f) = 0.46 (CH₂Cl₂—MeOH, 90:10.  8 Aldrich B1 H H b ¹HNMR(Methanol-d₄)8.13(dd, J=1.4, 4.6, 2H), 7.80(dd, J=2.0, 6.7, 2H),7.71(d, J=6.4, 1H),7.38-7.42(m, 2H),6.92(dd, J= 1.6, 4.7, 2H), 6.01(d,J=6.1, 1H); ES MS [M + H]⁺ = 296; TLC: R_(f) = 0.28(CH₂Cl₂— MeOH;90:10);  9 A1 B1 (CH₃)₃C— H b mp = 126-129° C.; ES MS [M + H]⁺ = 352;TLC: R_(f) = 0.62(CH₂Cl₂—MeOH, 90:10. 10 A1 B1 CH₃— Cl b ¹HNMR(Methanol-d₄)8.43(d, J=5.9, 2H), 7.93-7.96(m, 2H), 7.68-7.71(m, 2H),7.46(d, J=6.6, 2H), 2.51(s, 3H); ES MS [M + H]⁺ = 344. 11 A1 B1 —(CH₂)₄—b mp = 321-324° C.; ¹H NMR(DMSO-d₆) 9.35(s, 1H), 8.37(dd, J=1.4, 4.7,2H), 7.91 (d, J=8.9, 2H), 7.58(d, J=8.4, 2H), 7.02 (dd, J=1.5, 4.6, 2H),3.33(brs, 4H), 2.60 (brs, 2H), 1.77(brs, 4H); ES MS [M + H]⁺ = 350. 12Aldrich B1 H H c ¹H NMR(Methanol-d₄)8.26(dd, J=1.4, 4.7, 2H),8.11-8.16m, 1H), 7.84(d, J=6.2, 1H), 7.45-7.50(m, 2H), 7.04(dd, J=1.6,4.9, 2H), 6.11(d, J=6.1, 1H); ES MS [M + H]⁺= 314; TLC: R_(f) =0.40(CH₂Cl₂—MeOH, 90:10). 13 A1 B1 (CH₃)₃C— H c ¹HNMR(Methanol-d₄)8.25-8.27(m, 2H), 8.11-8.16(m, 1H), 7.46-7.48(m, 2H),7.03 (d, J=4.9, 2H), 6.15(s, 1H), 1.28(s, 9H);ES MS [M + H]⁺ = 370; TLC:R_(f) = 0.55(CH₂Cl₂— MeOH, 90:10). 14 A1 B1 —(CH₂)₄— c mp = 248-250° C.,ES MS [M +H]⁺ = 368; TLC: R_(f) = 0.56(CH₂Cl₂—MeOH, 90:10). 15 A1 B1—(CH₂)₅— c ¹H NMR(Methanol-d₄)8.78(d, J=6.6, 2H), 8.52(d, J=5.9, 1H),8.34(brs, 1H), 7.76(d, J=6.9, 2H), 7.42(d, J=6.1, 1H), 7.23-7.33 (m,1H), 7.11(brs, 1H), 6.51-6.58(m, 3H), 2.85-2.87(m, 2H), 2.60-2.63(m,1H), 2.31- 2.34(m, 2H), 1.42-1.75(m, 6H); ES MS [M + H]⁺ = 382. 16 A1 B1CH₃— H a mp = 254-256° C., TLC: R_(f) = 0.03(95:5 CH₂Cl₂/MeOH). 17 A7B16 CH₃— H e TLC: R_(f) = 0.23(6% MeOH/94% CH₂Cl₂); LC MS [M + H]⁺ =344; (3.37 min) 18 A7 B10 CH₃— H h TLC: R_(f) = 0.39(6% MeOH/94% CH₂Cl₂)LC MS [M + H]⁺ = 345; (3.37 min) 19 A7 B17 CH₃ H g TLC: R_(f) = 0.44(6%MeOH/94% CH₂Cl)₂ LC′MS[M + H]⁺360; (2.64 min) 20 A17 B7 Ph H c TLC:R_(f) = 0.26(4% MeOH/96% CH₂Cl₂); LCMS: ES[M + H]⁺ 390; (2.76 min) 21A18 B14 3-pyridyl H f TLC: R_(f) = 0.37(6% MeOH/94% CH₂Cl₂); LCMS: ESm/z 441(1.65 min) 22 A18 B5 3-pyridyl H b TLC: R_(f) = 0.35(4% MeOH/96%CH₂Cl₂); LCMS: ES[M + H]⁺ 373; (2.61 min) 23 A2 B5 4-pyridyl H b TLC:R_(f) = 0.13(4% MeOH/96% CH₂Cl₂); LCMS: ES[M + H]⁺ 373; (2.56 min) 24A17 B11 Ph H i TLC: R_(f) = 0.21(2% MeOH/98% CH₂Cl₂); LCMS: ES[M + H]⁺424; (2.75 min) 25 A2 B11 4-pyridyl H i TLC: R_(f) = 0.35(6% MeOH/94%CH₂Cl₂); LCMS: ES[M + H]⁺ 425; (2.60 min) 26 A7 B5 CH₃ H b LCMS: ES[M +H]⁺ 310; (3.53 min)

[0089] By selecting combinations of the appropriate Intermediates A1-A21with Intermediates B1-B17, a variety of products were prepared in likemanner and are described in Example 27-31.

Example 27

[0090] Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-{4-[(4-pyridinylmethyl)sulfanyl]phenyl}amine

[0091] Prepared in 34% yield from Intermediate A7 and B8: TLC (7% MeOHin CH₂Cl₂) R_(f) 0.36; MS (ES) 324 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 2.09 (s,3H), 4.12 (s, 2H), 5.87 (s, 1H), 6.33 (bs, 2H), 7.19 (d, J=8.5 Hz, 2H),7.23 (d, J=5.8 Hz, 2H), 7.64 (d, J=8.5 Hz, 2H), 8.43 (d, J=5.3 Hz, 2H),9.20 (bs, 1H).

Example 28 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-{4-[(4-pyridinylsulfanyl)methyl]phenyl}amine

[0092]

[0093] Prepared in 6% yield from Intermediate A7 and B18:TLC (7% MeOH inCH₂Cl₂) R_(f) 0.38; MS (ES) 342 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 2.06 (s, 3H),4.30 (s, 2H), 5.84 (s, 1H), 6.13 (bs, 2H), 7.27-7.31 (m, 4H), 7.63 (d,J=7.9 Hz, 2H), 8.33 (d, J=6.1 Hz, 2H), 8.99 (bs, 1H).

Example 29 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-{4-[2-(4-pyridinyl)ethyl]phenyl}amine

[0094]

[0095] Prepared in 30% yield from A7 and B6: TLC (8% MeOH in CH₂Cl₂)R_(f) 0.34; MS (ES) 306 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 2.06 (s, 3H),2.83-2.87 (m, 4H), 5.82 (s, 1H), 6.09 (bs, 2H), 7.07 (d, J=8.5 Hz, 2H),7.21 (d, J=5.8 Hz, 2H), 7.54 (d, J=8.5 Hz, 2H), 8.41 (d, J=6.2 Hz, 2H),8.87 (s, 1H).

Example 30 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-[4-(4-pyridinylsulfanyl)-3-(trifluoromethyl)phenyl]amine

[0096]

[0097] Prepared in 1.2% yield from A7 and B9: TLC (7% MeOH in CH₂Cl₂)R_(f) 0.39; MS (ES) 378 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 2.03 (s, 3H), 5.94(s, 1H), 6.33 (bs, 2H), 6.91 (d, J=6.5 Hz, 2H), 7.64 (d, J=8.9 Hz, 1H),8.19 (d, J=2.2 Hz, 1H), 8.33 (d, J=5.9 Hz, 2H), 8.37 (dd, J=2.1, 8.6 Hz,1H), 9.66 (s, 1H).

Example 31 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-[4-(5-isoquinolinyloxy)phenyl]amine

[0098]

[0099] Prepared in 30% yield from A7 and B10: TLC (6% MeOH in CH₂Cl₂)R_(f) 0.39; MS (ES) 344 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 2.08 (s, 3H), 5.85(s, 1H), 6.11 (bs, 2H), 7.02-7.08 (m, 3H), 7.58 (t, J=8.1 Hz, 1H), 7.74(d, J=8.6 Hz, 2H), 7.84 (d, J=8.2 Hz, 1H), 7.98 (d, J=5.8 Hz, 1H), 8.54(d, J=5.9 Hz, 1H), 9.03 (bs, 1H), 9.35 (bs, 1H).

Example 32 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-[3-fluoro-4-(5-isoquinolinyloxy)phenyl]amine

[0100]

[0101] A suspension of 2-amino4-chloro-6-methylpyrimidine (IntermediateA7, 0.14 g, 1.0 mmol), 3-fluoro-4-(5-isoquinolin-oxy)aniline(Intermediate B10, 0.25 g, 1.0 mmol), and HCl (0.1 mL) in H₂O (1.0 mL)was heated to 70° C. in a 5 mL reaction vial overnight. The reactionmixture was diluted with MeOH, treated with saturated NaHCO₃, and coatedon silica and purified by MPLC (Biotage) with 5% MeOH in CH₂Cl₂. Itafforded 52 mg of product (14% yield). TLC (6% MeOH/94% CH₂Cl₂) R_(f)0.45; MS (ES) 362 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 2.10 (s, 3H), 5.88 (s, 1H),6.29 (bs, 2H), 6.93 (d, J=7.9 Hz, 1H), 7.22 (t, J=8.9 Hz, 1H), 7.34 (dd,J=1.7, 8.9 Hz, 1H), 7.55 (t, J=8.1 Hz, 1H), 7.82 (d, J=8.3 Hz, 1H), 8.08(d, J=5.6 Hz, 1H), 8.21 (dd, J=2.6, 14.2 Hz, 1H), 8.58 (d, J=5.9 Hz,1H), 9.30 (bs, 1H), 9.36 (s, 1H).

[0102] Using the above-described method for Example 32, Examples 33-41were similarly prepared.

Example 33 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-[3,5-dichloro-4-(4-pyridinylsulfanyl)phenyl]amine

[0103]

[0104] Prepared in 10% yield from A7 and B14: TLC (5% MeOH in CH₂Cl₂)R_(f) 0.14; MS (ES) 378 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 2.14 (s, 3H), 5.92(s, 1H), 6.49 (bs, 2H), 6.93 (d, J=6.1 Hz, 2H), 8.13 (s, 2H), 8.35 (d,J=6.2 Hz, 2H), 9.63 (bs, 1H).

Example 34 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-[3,5-dichloro-4-(5-isoquinolinyloxy)phenyl]amine

[0105]

[0106] Prepared in 58% yield from A7 and B12: TLC (70% EtOAc/30%hexanes) R_(f) 0.18; MS (ES) 412 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 2.13 (s,3H), 5.89 (s, 1H), 6.38 (bs, 2H), 6.73 (d, J=7.6 Hz, 1H), 7.52 (t, J=7.9Hz, 1H), 7.82 (d, J=8.2 Hz, 1H), 8.05 (s, 2H), 8.16 (d, J=5.8 Hz, 1H),8.61 (d, J=5.9 Hz, 1H), 9.36 (s, 1H), 9.42 (s, 1H).

Example 35 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-[6-(4-pyridinylsulfanyl)-3-pyridinyl]amine

[0107]

[0108] Prepared in 16% yield from A7 and B13: TLC (6% MeOH in CH₂Cl₂)R_(f) 0.16; MS (ES) 311 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 2.11 (s, 3H), 5.91(s, 1H), 6.34 (bs, 2H), 7.15 (d, J=6.2 Hz, 2H), 7.48 (d, J=8.8 Hz, 1H),8.30 (dd, J=2.5, 8.4 Hz, 1H), 8.38 (d, J=6.1 Hz, 2H), 9.00 (d, J=2.4 Hz,1H), 9.45 (bs, 1H).

Example 36 Preparation ofN-(2-amino-6-phenyl-4-pyrimidinyl)-N-[4-(4-pyridinylsulfanyl)phenyl]amine

[0109]

[0110] Prepared in 65% yield from A17. TLC (4% MeOH in CH₂Cl₂) R_(f)0.22; MS (ES) 372 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 6.46 (bs, 2H), 6.55 (s,1H), 6.96 (d, J=4.8 Hz, 2H), 7.46-7.49 (m, 5H), 7.91-8.00 (m, 4H), 8.32(d, J=4.9 Hz, 2H), 9.57 (bs, 1H).

Example 37 Preparation ofN-[2-amino-6-(3-pyridinyl)-4-pyrimidinyl]-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0111]

[0112] Prepared in 45% yield. TLC (4% MeOH in CH₂Cl₂) R_(f) 0.27; MS(ES) 391 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 6.58 (s, 1H), 6.70 (bs, 2H), 6.99(d, J=6.4 Hz, 2H), 7.46-7.57 (m, 3H), 8.23-8.36 (m, 4H), 8.65 (d, J=4.4Hz, 1H), 9.09 (s, 1H), 9.86 (bs, 1H).

Example 38 Preparation ofN-[2-amino-6-(4-pyridinyl)-4-pyrimidinyl]-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0113]

[0114] Prepared in 22% yield from A2 and B7:. TLC (6% MeOH in CH₂Cl₂)R_(f) 0.32; MS (ES) 391 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 6.63 (s, 1H), 6.74(bs, 2H), 6.99 (d, J=5.8 Hz, 2H), 7.46-7.58 (m, 2H), 7.85 (d, J=5.8 Hz,2H), 8.31-8.36 (m, 3H), 6.70 (d, J=4.3 Hz, 2H), 9.92 (bs, 1H).

Example 39 Preparation ofN-[2-amino-6-(4-pyridinyl)-4-pyrimidinyl]-N-[3,5-dichloro-4-(4-pyridinylsulfanyl)phenyl]amine

[0115]

[0116] Prepared in 0.4% yield from A2 and B14: TLC (4% MeOH in CH₂Cl₂)R_(f) 0.15; ¹H-NMR (DMSO-d₆) δ 6.60 (s, 1H), 6.82 (bs, 2H), 6.95 (d,J=5.9 Hz, 2H), 7.85 (d, J=5.9 Hz, 2H), 8.18 (s, 2H), 8.36 (d, J=3.8 Hz,2H), 8.71 (d, J=4.7 Hz, 2H), 9.99 (bs, 1H).

Example 40 Preparation ofN-[2-amino-6-(3-pyridinyl)-4-pyrimidinyl]-N-[3-fluoro-4-(5-isoquinolinyloxy)phenyl]amine

[0117]

[0118] Prepared in 54% yield from A18 and B11: TLC (5% MeOH in CH₂Cl₂)R_(f) 0.34; MS (ES) 425 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 6.53 (s, 1H), 6.59(bs, 2H), 6.96 (d, J=7.4 Hz, 11H), 7.26 (t, J=9.6 Hz, 1H), 7.38-7.59 (m,3H), 7.83 (d, J=8.2 Hz, 1H), 8.09 (d, J=5.7 Hz, 1H), 8.23-8.31 (m, 2H),8.58-8.65 (m, 2H), 9.09 (bs, 1H), 9.37 (bs, 1H), 9.61 (bs, 1H).

Example 41 Preparation ofN-[2-amino-6-(2-pyridinyl)-4-pyrimidinyl]-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0119]

[0120] Prepared in 60% yield from A19 and B7: TLC (50% EtOAc/50%hexanes) R_(f) 0.14; MS (ES) 391 [M+H]⁺; ¹H-NMR (DMSO-d₆) δ 6.63 (bs,2H), 6.98 (d, J=6.6 Hz, 2H), 7.17 (s, 1H), 7.45-7.57 (m, 3H), 7.93 (dt,J=1.9, 7.7 Hz, 1H), 8.23-8.37 (m, 4H), 8.65-8.67 (m, 1H), 9.90 (bs, 1H).

Example 42 Preparation ofN-(2-amino-6-ethyl-4-pyrimidinyl)-N-[4-(4-pyridinylsulfanyl)phenyl]amine

[0121]

[0122] A mixture of Intermediate B5(50.6 mg, 0.250 mmol) and2-amino-4-ethyl-6-chloropyrimidine (Intermediate A8, 39.4 mg, 0.25 mmol)in 0.01 M aqueous HCl (500 μL) was refluxed for 6 h. The reaction wascooled to room temperature and the solvent was evaporated by vacuum. Theresidue was purified by reverse phase chromatography on a YMC Pack-pro®C18 column eluting with acetonitrile/H₂O (10:90-90:10 gradient) to givethe desired compound (13.0 mg, 0.040 mmol; 16% yield); mp=181-186° C.;ES MS [M+H]⁺=324; TLC (CH₂Cl₂—MeOH, 95:5); R_(f)=0.41.

Example 43 Preparation of ofN-(2-amino-6-chloro-4-pyrimidinyl)-N-[3-fluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0123]

[0124] 2-Amino-4,6 dichloropyrimidine (A21, 12 mmol) and3-fluoro-4-(4-pyridinylthio)-aniline (B7,12 mmol) were suspended inwater (150 mL) and treated with 10 drops of concentrated hydrochloricacid. The mixture was stirred at 100° C. overnight. The clear solutionwas then neutralized with ammonium hydroxide. The precipitated yellowproduct was filtered, washed with water, and purified by columnchromatography with 1-3% MeOH in CH₂Cl₂ to give the desired product as awhite solid (1.98 g, 47%).

Example 44 Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-[2,5-difluoro-4-(4-pyridinylsulfanyl)phenyl]amine

[0125]

[0126] The compound was prepared using a similar method used for thepreparation of Example 1 (described above) from A7 and B15: HPLC/MS:[M+H]⁺ 346.1 m/z. Retention time (HPLC/MS)=1.39 min.

Example 45 Preparation ofN-(2-amino-6-chloro-4-pyrimidinyl)-N-[3,5-dichloro-4-(4-pyridinylsulfanyl)phenyl]amine

[0127]

[0128] The compound was prepared using a similar method used for thepreparation of Example 43 (described above) from A21 and B14: HPLC/MS:[M+H]⁺ 399.0 m/z. Retention time (HPLC/MS)=3.02 min.

Example 46 Preparation ofN-(2-amino-6-chloro-4-pyrimidinyl)-N-[3-fluoro-4-(5-isoquinolinyloxy)phenyl]amine

[0129]

[0130] The product was prepared using a similar method used for thepreparation of Example 43 (described above) from A21 and B11: HPLC/MS:[M+H]⁺ 382.1 m/z. Retention time (HPLC/MS)=2.91 min.

Examples 47-49 Using procedures similar to the above examples and usingthe appropriate Intermediates A and B, the following examples wereprepared in like manner:

[0131] Example 47

Preparation ofN-(2-amino-6-methyl-4-pyrimidinyl)-N-[1-(4-pyridinyl)-1H-indol-5-yl]amine

[0132]

[0133] Prepared by reaction of Intermediate A7 and B1.

Example 48 Preparation ofN-(2-anilino-6-methyl-4-pyrimidinyl)-N-[1-(4-pyridinyl)-1H-indol-5-yl]amine

[0134]

[0135] Prepared by reaction of Intermediate A16 and B1.

Example 49 Preparation ofN-(2-anilino-6-methyl-4-pyrimidinyl)-N-[4-fluoro-3-(4-pyridinylsulfanyl)phenyl]amine

[0136]

[0137] Prepared by reaction of Intermediate A16 and B7.

Rho Kinase Biochemical Assay

[0138] ROCK-1 activity criteria: 0 no effect (<40% inhibition), 1 effect(>40% inhibition). The assay tests for inhibition of ROCK-1phosphorylation of MBP (Myelin Basic Protein). The reaction (100 μlfinal volume) is carried out in polypropylene 96-well plates in 50 mMHEPES buffer pH 7.5 containing 5 mM MgCl₂ and 1 mM DTT. For each well,gstROCK1 (0.25 μgs of BAYER DRT gstROCK1) is combined with MBP (1 μg) inreaction buffer (70 μL combined volume). Inhibitors (5 μL of 20×conc. in40% DMSO) are added to each well to give an 8 point dose response rangefrom 1.0 μM to 0.5 nM. The reaction is begun by adding 25 μL of ATP(4x=12 μM) in reaction buffer containing 0.8 μCi of ³³P gamma-ATP (4×)for a final concentration of 3 μM cold and 0.2 μCi hot ATP. Plates wereincubated for 1 hour at room temperature with the reaction being stoppedby addition of 7 μL of 1 N HCl. The radioactively labeled MBP wastransferred to P30 filtermats (EG&G Wallac), washed in 1% phosphoricacid followed by brief washes in water. The filtermats were then driedand the incorporation of ³³P detected by liquid scintillation counting.Background ³³P incorporation is determined by ROCK1 autophosphorylationwithout MBP. The data are expressed as percent inhibition: %inhibition=1−((cpm with inhibitor−background)/(cpm withoutinhibitor−background))*100.

[0139] The entire disclosure of all applications, patents andpublications, cited herein and of U.S. Provisional Application SerialNo. 60/349,986, filed Jan. 23, 2002 is incorporated by reference herein.

We claim:
 1. A compound of the formula


2. A method of treating an indication mediated by Rho-kinase, comprisingadministering a compound of claim
 1. 3. A process according to claim 1,wherein the host is a human.
 4. A method of treating hypertension,atherosclerosis, restenosis, cerebral ischemia, cerebral vasospasm,neuronal degeneration, spinal cord injury, cancer of the breast, colon,prostate, ovaries, brain or lung, thrombotic disorders, asthma,glaucoma, osteoporosis or erectile dysfunction, comprising administeringto host in need thereof a compound according to claim
 1. 5. A processaccording to claim 4, wherein the host is a human.